Stable flies are bona fide carriers of mastitis-associated bacteria

ABSTRACT Hematophagous Stomoxys (stable) fly populations in dairy barns are sustained by a constant availability of cattle hosts and manure, which serve as major reservoirs of both zoonotic and opportunistic bacterial pathogens. However, the composition of the Stomoxys fly microbiota, the mechanisms by which flies acquire their microbiome, and the ability of potentially pathogenic bacteria to colonize and persist in fly hosts remain to be investigated. Here, we longitudinally collected fly and manure samples from two connected dairy facilities. High throughput 16S rRNA gene amplicon sequencing was then used to characterize and compare bacterial communities present on or within flies and in manure collected from the same facility, while culture-dependent methods were used to verify the viability of clinically relevant bacteria. Bacterial alpha diversity was overall higher in manure samples as compared to fly samples, with manure-associated bacterial communities being dominated by members of the Bacteroidales, Eubacteriales, and Oscillospirales. In contrast, flies harbored relatively low-complexity communities dominated by members of the Enterobacterales, Staphylococcales, and Lactobacillales. Clinically relevant bacterial strains, including Escherichia spp. and other taxa associated with mastitic cows housed in the same facilities, were detected in paired fly and manure samples but exhibited dramatically elevated abundances in fly samples as compared to manure samples. Viable colonies of Escherichia, Klebsiella, and Staphylococcus spp. were also readily isolated from fly samples, confirming that flies harbor culturable mastitis-associated bacteria. This study identifies biting flies as bona fide carriers of opportunistically pathogenic bacterial taxa on dairy farms. IMPORTANCE Disease prevention on dairy farms has significant implications for cattle health, food security, and zoonosis. Of particular importance is the control of bovine mastitis, which can be caused by diverse bacteria, including Klebsiella, Escherichia coli, Streptococcus, and Staphylococcus spp. Despite being one of the most significant and costly cattle diseases worldwide, the epidemiology of bovine mastitis is not well understood. This study provides parallel culture-independent and culture-dependent evidence to support the carriage of opportunistically pathogenic bacteria by Stomoxys flies on dairy farms. We further show that the fly microbiota is enriched in clinically relevant taxa—the vast majority of which can be traced to the manure habitats in which flies breed. Altogether, our results identify biting flies as underrecognized carriers of bacterial taxa associated with environmental bovine mastitis and other opportunistic infections in vertebrates and offer important insights into mechanisms of microbial acquisition by these and other medically important insects.

Points are colored by sample source (internal fly samples, yellow; external fly samples, blue; manure samples, red), with each point representing one DCC-derived manure or fly sample community.PERMANOVA identified significant clustering by sample source for both sampling dates for which there were enough samples to compare communities (Jul15: P = 0.008, R 2 = 0.43; Jul22: P = 0.007, R 2 = 0.41).ASV taxonomy is shown at the genus level.Taxa highlighted in red were identified as commonly shared ASVs in both facilities.
Fig. S4PCoA of Bray-Curtis dissimilarities of community relative abundances, by sampling date.Points are colored by sample source (internal fly samples, yellow; external fly samples, blue; manure samples, red), with each point representing one DCC-derived manure or fly sample community.PERMANOVA identified significant clustering by sample source for both sampling dates for which there were enough samples to compare communities (Jul15: P = 0.008, R 2 = 0.43; Jul22: P = 0.007, R 2 = 0.41).
Fig. S5PCoA of Bray-Curtis dissimilarities of community relative abundances, colored by sampling location (see Figure1for more information).Each point represents one Arlington-(left) or DCC-derived manure (top) or fly (bottom) sample community.PERMANOVA identified significant clustering by sampling location for both Arlington-(manure: P = 0.001, R 2 = 0.21; fly: P = 0.003, R 2 = 0.03) and DCC-derived samples (manure: P = 0.006, R 2 = 0.11).However, significant post hoc pairwise comparisons were only identified for Arlington-and DCC-derived manure samples, including those between samples collected from the Arlington sick pen and other sampling locations at the same facility (P < 0.05).No significant pairwise comparisons were identified for Arlington-derived fly samples (P > 0.05).

Fig. S14
Fig. S14Relative abundance of commonly shared ASVs in Arlington-derived fly and manure samples, by sampling location (x-axis; see Figure1for more information).Libraries derived from samples collected from the same sample source at a given sampling location were pooled across sampling dates for the bar graphs presented.Colored bars present the proportion of sequencing reads assigned to a given ASV.ASV taxonomy is shown at the genus level.Taxa highlighted in red were identified as commonly shared ASVs in both facilities.

Fig. S15
Fig. S15 Relative abundance of commonly shared ASVs in DCC-derived fly and manure samples, by sampling date.Libraries derived from the same sample source on a given sampling date were pooled across sampling locations for the bar graphs presented.Colored bars present the proportion of sequencing reads assigned to a given ASV.ASV taxonomy is shown at the genus level.Taxa highlighted in red were identified as commonly shared ASVs in both facilities.

Fig. S16
Fig. S16 Relative abundance of commonly shared ASVs in DCC-derived manure samples, by sampling location (x-axis; see Figure 1 for more information).Libraries derived from samples collected at a given sampling location were pooled across sampling dates for the bar graphs presented.Colored bars present the proportion of sequencing reads assigned to a given ASV.ASV taxonomy is shown at the genus level.Taxa highlighted in red were identified as commonly shared ASVs in both facilities.